PROGRESSIVE LINEAGE ANALYSIS BY CELL SORTING AND CULTURE IDENTIFIES FLK1(-CADHERIN(+) CELLS AT A DIVERGING POINT OF ENDOTHELIAL AND HEMATOPOIETIC LINEAGES()VE)

Totipotent murine ES cells have an enormous potential for the study of cell specification. Here we demonstrate that ES cells can differentia te to hemopoietic cells through the proximal lateral mesoderm, merely upon culturing in type TV collagen-coated dishes. Separation of the Fl k1(+) mesoderm from other cell lineages was critical for hemopoietic c ell differentiation, whereas formation of the embryoid body was not. S ince the two-dimensionally spreading cells can be monitored easily in real time, this culture system will greatly facilitate the study of th e mechanisms involved in the cell specification to mesoderm, endotheli al, and hemopoietic cells. In the culture of ES cells, however, lineag es and stages of differentiating cells can only be defined by their ow n characteristics. We showed that a combination of monoclonal antibodi es against E-cadherin, Flk1/KDR, PDGF receptor alpha, VE-cadherin, CD4 5 and Ter119 was sufficient to define most intermediate stages during differentiation of ES cells to blood cells. Using this culture system and surface markers,we determined the following order for blood cell d ifferentiation: ES cell (E-cadherin(+)Flk1(-)PDGFR alpha(-)), proximal lateral mesoderm (E-cadherin(-)Flk1(+)VE-cadherin(-)), progenitor wit h hemoangiogenic potential (Flk1(+)VE-cadherin(+)CD45(-)), hemopoietic progenitor (CD45(+)c-Kit(+)) and mature blood cells (c-Kit(-)CD45(+) or Ter119(+)), though direct differentiation of blood cells from the F lk1(+)VE-cadherin(-) stage cannot be ruled out. Not only the VE-cadher in(+)CD45(-) population generated from ES cells but also those directl y sorted from the yolk sac of 9.5 dpc embryos have a potential to give rise to hemopoietic cells. Progenitors with hemoangiogenic potential were identified in both the Flk1(+)VE-cadherin(-) and Flk1(+)VE-cadher in(+) populations by the single cell deposition experiment. This line of evidence implicates Flk1(+)VE-cadherin(+) cells as a diverging poin t of hemopoietic and endothelial cell lineages.